Gene-based and Viral-based Immunotherapies for Glioblastoma Free

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- Colleagues and friends, thank you for joining us for another session of, "The virtual operating room," from Neurosurgical Atlas. My name is Aaron Cohen. Our guest today is Dr. Nino Chiocca from Brigham Women's Neurosurgery, he's the department chair there. He's also the Harvey Cushing Professor of Neurosurgery. Nino has been one of the most instrumental neurosurgeons of our time. His basic science research, his work on gliomas, his technical skills are unparalleled. Nino, it's truly an honor, having you with us today. There's been significant interest about immunotherapy, treatments for glioblastoma and your lecture today about gene-based and viral based immunotherapies for this tumor will be really important and timely and I really appreciate it and look forward to learning from you. So please go ahead and thank you.

- Thank you Dr. Cohen-Gadol, and I'll call you Aaron. It is a real privilege to be here today with you. I'm going to talk about a clinical research program that I've been involved with for a while, related to glioblastoma, an incurable cancer that still affects over 20,000 patients in the United States every year. And really the lesson here is how you can use your clinical research program, to do something for patient during surgery. So here we're implementing an immunotherapy, during surgery and so you are complimenting both your clinical interest and your research interests in the operating room. So, let me get the next screen. So these are my disclosures and financial support. So we'll just start off with, what happens with a patient with glioblastoma. The title says 2022 and I can assure you, nothing has changed in 2023. And so this is how a patient can present, this was a patient of mine. He was a a Afghanistan war veteran and he presents with speech anomalies or speech issues, because of this gadolinium enhancing lesion, neo broca's area. And we took him to surgery to try to get what's called a total surgical resection. This is the gadolinium SMRI scan, there's a little blood in the cavity, if you look at the T1 one there, that's blood and not enhancement, but you try to get what's called a gross total resection and then the typical course, is to follow this chemo radiation protocol for six weeks and then followed by adjuvant temozolomide, with or without tumor treating fields and this is called the Stupp Regimen. But invariably most of these patients recur and usually recur historically, the recurrence rate's within six to nine months and this is what happened to this patient. As you can see, there is a recurrence in the tumor cavity and at that time, at the time of recurrence, all bets are off. You can do more surgery, which is what we did in this case and try to resect the recurrence and then most patients can go onto a clinical trial, 'cause there's really no approved therapy for this recurrence. They can go on off-label chemo, but invariably what happens, is that the recurrence occurs again and here's a second recurrence for this patient and now the tumor has gone through the corpus callosum to the other side. Now it's gone into areas of the brain that are completely unresectable and unfortunately passed away, shortly after this. So the median survival time from initial diagnosis is anywhere between 15 to 20 months, but this really varies greatly based on genetic and demographic markers and we now know that if you have a mutation in the gene for IDH, you actually don't even call it the glioblastoma anymore, you call it an IDH mutant grade for astrocytoma, because we know that those patients live longer, regardless of what you do to them. If you have a methylation of the MGMT promoter, we know that those patients also seem to do a little bit better, because those tumors can be more chemo and radiation sensitive. Ultimately, all patients end up recurring, unfortunately dying. There's also some variation in this, because this median survival time formulation diagnosis is really based on clinical trial data and in clinical trial, there are strict eligibility criteria. I'm actually fairly amazed, when you look at a registry data, these are the registries that collects all patients from the the United States and registry databases, if you look at the median survival for glioblastoma patients in registry, so this includes patients with even the worst demographic and imaging characteristics like multifocal GBM or GBM that are deep, the median survival time is still eight months and has really not changed since the last century. We didn't know a lot about these tumors. In fact, this tumor was the first tumor to be sequenced by the TCGA group back in the mid 2000s, so we know a lot about the genetic abnormalities of these tumors, we know a lot of the mutations of these tumors, there are a lot of targets, but all the clinical trial target therapies have failed. There was some excitement in the 2000s, 2010s, because angiogenesis and drugs against angiogenic messages in the tumor were out like bevacizumab. But even the antigenic therapies have not been successful. And finally, with the advent of immunotherapy and immunotherapies are now getting FDA approved, left and right, for a number of solid tumors, there's a lot of excitement about using immunotherapy in clinical trials for these tumors, but they also have not been successful. So the next part of my talk is really focused on immunotherapy and actually surgical immunotherapy, how we can do immunotherapy during surgery, in clinical trials. So the problem with glioblastoma and why immunotherapies have not been successful, has been the fact that this is the prototypical immunologically cold, also called lymphocyte depleted or immune deserted tumor. What do I mean by that? Well the sine qua non for immunotherapy to work is based on the fact that you need to have a particular cell, called a cytotoxic CD8+ T cell, coming to direct contact with a tumor cell and in this contact, the T cell basically releases enzymes called perforin granzyme B and others, to destroy the tumor cell and then the T cell can go on and destroy other tumor cells. So there needs to be contact. The problem with glioblastoma, is when you analyze the tissue, you see very few T cells in the tissue. In fact, the tumor is actually full of other cells in the immune system called myeloid-derived suppressor cells, that actually create an environment of immunosuppression and T cells cannot live and do not penetrate the tumor. And therefore, whenever you try to do an immunotherapy, particularly stem chemotherapies, it is really impossible for these T cells to get into the tumor, even if they're activated and that's been the problem with the immune checkpoint therapies that have failed the GBM, they just have not been able to get the T cells, even if activated, to get into the tumor. This is very different from melanoma. Melanoma is recorded as a warm tumor, they're the words. There are a lot of T cells already in the tumor and therefore when you activate by immune checkpoint inhibitors, those T cells are already there and they can do their own job. So what we have been doing and what I want to convince you of, and this is sort of like the slide, this is what we're doing, which is at the time of surgery, instead of getting systemic immunotherapy, we wanna give patients intratumoral immunotherapy to get more T cells to react. And so here on the left, what you see is your typical glioblastoma, that has lots of myeloid-derived suppressor cells and here almost like saving the tumor from the T cells. But what we'd want to do, is incite to administer things that will change this balance between this immunosuppressive myeloid sub-compartment and these immunoreactive T cells, so that you can get the opposite. Many more immunoreactive green T cells in here and less of the blue immunosuppressive myeloid cells. And the way we do this is by administering, and now let's first talk about this powerful cytokine gene therapy and the second part of my talk will be administering a powerful oncolytic virus. So really what this is, is a gene therapy approach I call the surgical immuno-gene therapy and really what we wanna do, is express a powerful immuno-stimulant in tumor and you don't need to express this in all tumor cells, you're just trying to get a few tumor cells, to make this immuno-stimulant like a powerful cytokine to rev up the immune system and get more T-cells into your tumor. And the way we deliver this in surgery is by using what's called an adenoviral vector. This is just a vector, it's a virus, but it's been disabled, so it really does not make any virus, it just delivers the gene into the tumor cell. And so again, you know, for the last 20 years, I've been doing clinical trials of technologies like this. We have few clinical trials, at the time, of newly diagnosed glioblastoma that have been published, but really most of the trials we do, is at the time of recurrence, when the patient shows up, either first or second recurrence, or sometimes even a third recurrence. So the first trial I'm gonna talk to you about, is delivering a powerful cytokine, this is a cytokine for interleukin 12. At the time of surgical reception of the tumor, where we peritumorally inject, after we dissected the tumor, this adenoviral vector that makes interleukin 12 and this was published in Science Translational Medicine 2019. So why do we want to deliver interleukin 12, IL-12? IL-12 was described in the late 1980s and upon its discovery, it was pretty much, scientists figured out it was a very mass regulator of the immune system. It stimulates several types of immune cells, to become more anti-tumor like. It makes CD8+ T cells makes lots of interferon gamma, interferon gamma is a downstream modulator of IL-12, so it really is what gets the immune system revved up, IL-12 is a signal and then cells like T cells, NK cells, CD4 cells start making lots interferon gamma. And we now know that interferon gamma is what really drives the immune system to reject the tumor. So IL-12 was sort of this powerful regulator of interferon gamma, as well as of other immune activating molecules. However, when IL-12 was first discovered, it was first applied in oncology as a recombinant protein, where patients got systemic IL-12. But this was way too toxic. It was too powerful, those cytokines and patients got what's called a cytokine release syndrome, with kidney and liver failure and therefore those trials were pretty much abandoned. There was a thought of trying to deliver the protein intratumorally, to get rid of the systemic effect, but the problem is that with common protein, the half-life is too short, so it did not hang out in the tumor and just did not persist in the tumor. Although I should say now, there are preclinical experiments, where people are using parts of the IL-12 recombinant protein, that then are targeted to antibodies or conjugates, that will keep the protein in the tumor itself, but none of those have seen a clinical trial yet. The solution therefore to try to get rid of this, is to deliver this intratumorally by using a gene therapy modality. Where you deliver the gene, you infect cells in the tumor and now you make IL-12. A second thing that we did, well the company that I worked with did, was to try to make this even safer, by putting the IL-12 gene under control of what's called the RheoSwitch component. This is a promoter element. Promoters are what turns genes on and off and this promoter element, actually, is inactive. So when you first deliver this vector with IL-12, IL-12 is not getting made in the cell, because this promoter is inactive. It's off. So the switch gets turned on, the promoter gets turned on, once you give the patient, or the animal initially, a small activator ligand. The ligand is actually called Veledimex, VDX and this turns down the promoter and now you start making IL-12. So this is a small video, cartoon that explains how this works.

- [Presenter] Injected with Ad-RTS-hIL 12, a genetically engineered non-replicating adenoviral vector. It implants genetic code for IL-12 and the RheoSwitch, to regulate IL-12 expression. Non-local expression of IL-12, The patient takes a capsule called Veledimex. Veledimex controls the amount of IL-12 expressed. IL-12 recruits an immune response, such as T-cells, directly to the tumor environment.

- Okay so that is the idea behind this. So we instituted a phase one study, to see whether this gene therapy could be used in a safe manner, in patients with current or progressive glioblastoma. The centers that accrued were the Brigham, Cedars-Sinai, University of Chicago, University of California, San Francisco, Northwestern. So these are again, with patients of recurrent GBM, they were scheduled for a standard of care resection. Now three hours before the resection, we gave them the activator ligand, the drug Veledimex. The reason being, is that we wanted to be able to assess, whether the drug got into the tumor, did it get across the blood band barrier and did it get into the tumor? We then took patients to surgery, resected the tumor, tried to do what's called a gross cell resection and then free-hand injected this adenoviral vector with this promoter, the RheoSwitch promoter that's off and that supposedly drives IL-12. And then for 14 days after surgery, the patient took this drug Veledimex, to turn on this promoter, so the patient would make IL-12 in the tumor. So this was a dose escalation trial, trying to find a dose that was safe. We started with 20 milligrams of Veledimex, to turn on this promoter and this seemed to be safe, well tolerated by patients, in fact patients were able to complete the entire 14 days of treatment and then we escalate to 40 milligrams. Now patients did not tolerate this. They did get what's called, a cytokine release syndrome, because of too much alcohol being made and getting spilled out in the blood. In fact, most patients were not able to complete this course, we had to stop the drug, because of toxicity. So we tried to do this at 30 milligrams, but the same thing happened. So finally we came up with 20 milligrams being the safe dose or what's called the maximum tolerated dose, which is what you're trying to find in a phase one trial. What is the maximum tolerate dose you can give to patients? And again, this was published in Science Translational Medicine 2019, if you wanna look at the paper. So we had two scientific questions we wanted to answer in this trial, outside of the safety. First of all, does the Veledimex ligand actually cross the BBB, the blood brain barrier and gets into the tumor? And number two, if it gets into the tumor, does it really turn on the transcription, the gene, after it was injected into the resected glioblastoma cavity? So this slide tries to answer both of those questions, okay? So we actually have here, we're measuring the plasma concentrations of patients that got the 20 milligrams of drug, which was well tolerated, or the 30 or 40 milligram of drug that was not tolerated. These are the peak plasma concentrations and at the time of surgery, if you look at the drug and tumor, you do see that there's about 40% of the drug ends up being in tumor and these are the peak tumor levels, at the 20 milligram level, 30 milligrams, 40 milligrams. So yes, there seems to be a lot of drug that goes from the plasma into the tumor. It does cross the blood brain barrier, it does get into the tumor. The second question we asked again was, "Well if it's there, does it control transcription of the gene?" So here we're looking at interleukin 12 assays from the blood of patients. And what you can see here, based on, there's no IL-12, that you really can detect in the patient's blood. However, as you increase the amounts of drug and if you give, for example 20 milligrams, now you see that there is IL-12 in the blood, being detected and the more drug you give, the more IL-12 gets into the blood. Actually these are fairly massive quantities of IL-12, which is why patient probably did not tolerate this well. If you stop the drug within 12 to 24 hours, you can see that IL-12 levels return back to baseline. And then finally, we also asked, "IL-12 is the upstream modulator, but really what IL-12 is supposed to do is turn on expression of interferon gamma, from all of the immune cells I discussed before. But we also assert interferon gamma levels in the blood of patients. And again, without the drug, there's no interferon gamma that you can really detect in blood, but as soon as you give the drug, which you can start seeing, is there's interferon gamma, that spills out in the blood of patients and at the safe dose and there's even more at the doses that were not well tolerated. And if you stop the drug, what you can see is, the interferon gamma levels go back to baseline. So the answer to that question is, "Yes." It crossed the blood brain barrier, it turns down transcription to genes and this is reversible. As soon as you stop the drug, gene transcription goes away. So even though this was a phase one trial and it's not designed as a phase one trial, does not have the power to look at survival, we want to see clearly, is there any evidence that patients seem to survive more. So here we're just looking at the survival curves and just look at the blue curves. These are the patients that underwent the treatment with 20 milligrams. In other words, they completed the entire course of treatment, as you can see here. And what you can see, is the median raw survival's about 12.7 months. Now, the historical, and again there's always bias when you compare to historical data, but the historical data for survival from recurrent GBM is six to nine months. So this seemed to be encouraging. We also saw, and I'm not showing that curve, a subset of patients who had low dose dexamethasone or steroids, being given, as they were taking that 14 days of Veledimex and if you took less than 20 milligrams of cumulative dexamethasone, during that 14 day period of time, the medial survival of those patients was almost 19 months. So perhaps steroids had a negative impact on the survival of those patients. So next question we ask, is when patients recurred, again after this gene therapy, is there any evidence from tumor that you could resect, that there was an immuno-stimulation? And so here, we took some tumors from patients and you can use these multiplex immunofluorescence analyses and what you can see, in a lot of the patients that got the gene therapy, you actually get these, lots of immune infiltrates and these immune infiltrates are composed primarily, of T cells, as shown here and you can even quantify this. So after treatment, in fact, even four to six months after treatment, of these glioblastomas, what you can see, in each one of these doctors, the patients, there's only three patients per sample here. But what you can see, a baseline, there's very few lymphocytes, but as soon as, even four to six months after gene therapy treatment, you can see an increase in lymphocytes in these tumors and these are cytotoxic. But interestingly, they also expressed the immune checkpoint inhibitor of PD1. Which may be a reason, perhaps, why these tumors do recur, even though there's lots of lymphocytes in these tumors, they do end up recurring, because maybe these lymphocytes are PD1+, therefore they're exhausted. And in fact the ligand for PD1 is called PD-L 1, that's expressed in tumor cells, as well as other cells in the microenvironment and that was also over expressed. Basically, this is still an immuno-stimulatory environment, there's lots of T cells that may be exhausted, but it's immuno-stimulatory, because you can still measure interferon gamma, even in four to six months and these interferon gamma levels in the tumor, are elevated, compared to baseline, is shown here and elevated quite a bit, as you can see here, from these Y axis. And so what this told us, is that just one application, doing surgery of this gene therapy does change the tumor microenvironment in one that's now full of lymphocytes, even though they may be exhausted and is full of interferon gamma. So this has turned the glioblastoma from a lymphocyte depleting immune deserted tumor, into one that's more propitious or favorable for immunotherapy. So based on the PD1 experiments, or the PD1 results, we said, "Ah, ha!" So what we should do next, is do a combination trial of this gene therapy with an immune checkpoint inhibitor, delivered systemically and we did a phase one with nivolumab, that was published in 2021, but also we started a phase two, that actually is finished and the manuscript is in preparation. We're seconding a new checkpoint inhibitor called Libtayo. But, so sorry, before I say, "But," so this is the trial design, you would give the immune checkpoint inhibitor, before surgery, wait seven days, give the drug, take the patient to surgery, resect the tumor, then give again the drug to activate IL-12 and then the patient underwent the immune checkpoint infusion, every two weeks. But and this is a but, the surprise was, that the drug did not improve the IL-12 gene immunotherapy. We published this and again, these are the Kaplan-Meier survival curves of patients and in green, it shows the patients that only got the IL-12 gene therapy, this is their survival, shown here. And then, if you look at patient that got one immune checkpoint in the phase one in blue or the phase two in red, these survival curves are completely the same. So the addition of the immune checkpoint inhibitors, did not improve IL-12 gene immunotherapy, which was a surprise. So we tried to query tissue. So in other words, after the surgery, when patients recurred, we tried to get tumors back. And when you do that and you do these multiplex immuno fluorescence analysis, to look at what type of immune cells are in there and these are just results from two patients and I wanna first show you these two patients, patient 123, patient 128 and each one of these rows is really a section of a slide, where you are looking at the expression for example, PD1. And what you can see in this particular patient, is that before the gene therapy and the immunotherapies, you get some PD1 expression, but afterwards, you get much less. So yeah, clearly, when you give this immune checkpoint inhibitor, you stop PD1 expression in the tumor. But I'm gonna then show you the red curve, red arrows, you also stop infiltration of T cells in the tumor. Here in this particular case are the CD8+ T cells and what you can see is, there some T cell in the tumor before surgery, but after surgery and nivolumab, there's many less T-cells, which was the opposite of what we had seen in the IL-12 monotherapy trial. So somehow, when you give Anti-PD1, we're not improving the IL-12 gene immunotherapy and maybe that, even though you're downgrading PD1, it makes these T cells less able to get into the tumor. Now I don't know the answer to this, why that is so. But we start exploring and these are just a few slides about research. So these are really lab slides and it's now being clear that it's not just PD1 that's an important immune checkpoint inhibitor in tumors, but there's a plethora of other ones. Here's a T cell when it encounters a tumor cell and these are all the immune checkpoints that can be stimulated or activated or down-regulated. And so we started to reason then maybe, like everything, when you target just one molecule, the tumor finds ways to evade that targeting and goes and turns on other immune checkpoint signals. And so we became interested in trying to find a common vulnerability, something that's upstream of a lot of immune checkpoint signals or immunomodulation pathways and we started working on what's called, long non-coding RNAs. Long non-coding RNAs are basically RNAs that are made, but do not make protein, in other words they don't code for protein. And in fact, more than 97% of these RNAs in the cell do not make protein and it's now being realized, that these RNAs are actually regulatory RNAs, that regulate when messenger RNAs, the ones that make protein are made, they regulate the timeframe, they regulate the localization, so they are really key elements of the regulatory network of gene expression. But it's also been known, that long non-coding RNA seem to play a key role in immunomodulation. And in fact, one of our junior faculty, who when he was a postdoc in my lab, started working on this non-coding RNA called INCR1 and the reason being is, that he showed the INCR1 is highly stimulated, as glioblastoma tried to immuno-evade an immune stress. And if you down-regulate INCR1, you turn off, as shown here, all these immuno-evasive pathways, including immunosuppress checkpoints, IDO1, FAS, PD-L1, these immunosuppressive enzymes and signaling pathways, immunosuppressive cytokines. You basically go from red to blue, blue is low and compare that, to what happens if you down-regulate, just one immune checkpoint, like PD-L1, only the PD-L1 pathway goes down, everything else stays red. So this again shows, that we found this common vulnerability, upstream of all these immuno-evasive pathways and if you knock that common vulnerability down, you can make glioblastoma cells much more immuno-sensitive and this, kind of, cartoon explains it, I'm not gonna go through it, but this paper was published and you can read it if you're interested. And this, Marco Menino did all these studies and this is just an animal model, showing that if you gave a single infusion of CAR-T cells, in this animal model, if you just give a single infusion and actually a low dose infusion, there's really no effect of the CAR-T cells, you need to really give many more CAR-T cells for this to happen. But if you knock down this common vulnerability, the INCR1 non-coding RNA, all a sudden you can make this low dose of CAR-T cells work really well, regarding the growth of this tumor, as shown here. So we're now interested in trying to pursue a clinical trial, where we're gonna inhibit INCR1, at the time of immunotherapy, to see if we can make tumor cells and GBM cells more immuno-sensitive and hopefully that will be performed soon. This is just another experiment, but these are what are called glioblastoma organoids, they're grown in culture, but they're growing in culture together with T cells. So you can see these little dots here are T cells and T cells, they're not activated, so they don't have interferon gamma, IL-12, really don't do anything to these organisms, they just hang around. But as soon as you activate them with IL-12 for example, these T cells will start attacking the glioblastoma and what you can see is, that this is much smaller than this, but if you knock out INCR1, the attack is much more vigorous and you can see that this organ, really just disappears from the culture and it gets completely strummed off by these T cells. This works much better, than if you just use a single target PD1 or anti-PD1 antibody, as shown here. So the first part of the talk, what I tried to show you, is that IL-12 gene delivery using adenoviral vector shows encouraging biologic and immunologic activity, in recurring glioblastoma patient. There's also some encouraging survival in patients. I did not show you the slides, but when we look at it, if the patient was on high-dose dexamethasone inhibited effect. Post-injection glioblastomas do show inflammatory infiltrates. But the question is, when these tumors seem to be recurring, is the evasion due to immune checkpoint signaling? We do see up-regulation of the PD1, PDL1 signaling pathway, but when we did a clinical trial combining against anti-PD1, we showed no improvement to IL-12 gene therapy. And so the way out of this was, is to try to find a broader manner to disrupt immuno-evasion, immune checkpoint signaling and really what we wanted to do next, is try to target the INCR1 long non-coding-RNA, using your small molecules on antisense and hopefully that trial will start in the relative future. So the funding, the collaborators, for this part of the talk are shown here, Marco Mineo did all the INCR1 experiments, but this was funded through a program project grant and also Ziopharm Oncology, which is now called Alaunos, was the sponsor of the trial. But these were the monotherapy clinical trial sites, with the PIs shown here. So for the second part of the talk, I'm gonna talk about another modality and again as I said, we're still trying to change the immuno-microenvironment of the tumor. I discussed it, dealing with IL-12, but now I'm gonna discuss it, dealing with an oncolytic virus. What is an oncolytic virus? So unlike the gene therapy approach, where you're using the virus, but it's just a vector, so it's not making any viral protein, here we're actually making a virus, that actually can make copies of itself, it can replicate and as it replicates its spreads the infection throughout the tumor. So it already has three modes of anti-tumor effects. The first one is a direct cytotoxic effect, the virus infects the tumor cell and it kills it. The second mode, is in this process, you're making a lot of inflammatory molecules and this stimulates macrophages, neutrophils and lots of components to the immune system interferons, which tend to be anti-tumor. But lastly, and this is what we're all trying to achieve, is we're trying to get T cells into the tumor, called in by this inflammatory response and having these T cells, not only recognized through their T-cell receptor viral antigen, but then reeducate them to also recognize tumor antigens, so that you can get a long-term adaptive vaccination effect against a glioblastoma. So that is the entire idea, behind the use of oncolytic viral therapy and really, I think that the success will be with number three. Can you get enough of this and can you get this in a durable fashion, to really vaccinate yourself against the GBM? So there's been a lot of excitement about this in the last few years, with some high profile papers and there was a poliovirus trial done at Duke, where they saw a tail of treatment responders and this you know, was published in the New England Journal of Medicine and now in the pediatric glioma population, there have been two New England Journal of Medicine papers, one using an oncolytic herpes virus, another one using an oncolytic adenovirus for both high-grade gliomas as well as DIPGs. And then, more recently, there was a trial in Japan of a different oncolytic herpes virus called G47 Delta, each one of these viruses, engineered a little bit differently, okay? So G47 Delta showed increased survival in patient with GBM. So this basically got FDA approval by the Japanese FDA and more recently, last month, there was a trial published by the Group of Anderson and Gelareh Zadeh from Toronto of using an oncolytic adenovirus, same one they used for the DIPG paper here, in combination with pembrolizumab. I wanna just briefly discuss this Japanese paper, because it did get to a Japanese FDA approval. So everything I'm telling you here, with these oncolytic viruses is they're usually delivered, therotatroly in unresected tumors. So these are tumors that are not resected, you just give a one time injection. But for the first time in Japan, what they did is, they did a longitudinal injection. So patients were under, as well, stereotactic inoculation of this oncolytic virus, up to six times, almost like chemotherapy, we give it multiple times. So the first time, they were able to show you can actually do this six times in a row, in patients. With my postdoc John Christie, we wrote a news and views article on this. But basically here is the virus, it was injected and then over a period of of four to five months, the patient they went further injections stereo-tactically and the nice thing about this trial is they actually got biopsies, so they could logically track what's happening to immune cells by using the chemistry in these tumors. And what you can see is, the more virus, or the more time you gave this virus, the more the increase in CD8+ T-cells, C4+ T cells, they usually are anti-tumor cells. And what got this trial approved by the Japanese FDA, is that they said, "You know at 12 months, the landmark survival of 12 months, only 15% of GBM patients were recurrence, a hundred percent of GBM are alive at 12 months." They used the data. In this trial there was 84% survivorship. So this is what got this approved by the FDA and then at two years, it was a disease stabilization in a large proportion of patients in the trial. So several years ago, in my laboratory, we engineered a different type of oncolytic herpes virus, this is the name for it, it's rQNestin34.5v.2. This has now been licensed to a company and they've renamed it CAN-3110, which is probably easier to say. So as you see through slides, we use CAN-3110, rQNestin, interchangeably. But the difference in this virus, compared to the other ones in clinical trials, is that we re-engineered it and the key here is, we engineered this virus to still express what's called the ICP34.5 gene. So what is ICP34.5? ICP34.5 has been removed from all the oncolytic herpes viruses in clinical trials, because it is the major neurovirus gene from herpes. This is what makes herpes encephalitis happen. This is what makes herpes meningitis happen. So if you take out this gene, those side effects don't happen, if you get a wild-type herpes infection in your brain, okay? The problem is, when you take out this gene, these viruses don't replicate very well. In other words, they don't infect and spread the infection really well and that's been shown over and over again. And so, the invention of what we did in my lab, was to say look, "Let's just put back ICP34.5 into the virus." 'Cause now the virus can be more potent, but try to restrict its expression, by using a glioma promoter. Promoter that's expressed only in gliomas in the brain, which is the promoter for nestin. Nestin is a marker for glioma stem cells, it's highly expressed in glioblastoma, but the adult human brain does not have nestin expression. So the idea here is, you would have a virus that when it got into the brain, it would replicate, infect, and inflame the tumor. But if it got into the brain surrounding the tumor, it would do nothing, okay? So we're trying to turn the virus, from one that's causing encephalitis into one that causes what I say GBM-itis or glioma inflammation, okay? And the nice thing about herpes, using all the oncolytic herpes, is that, first of all, it can be very safe, because if you worry about a side effect, you can turn off the virus, by using antiherpetic drugs, like valacyclovir, acyclovir. So we actually have a way of drugging and getting the virus to shut off. So our animal experiments looked great and so I decided back, I'm not sure when, to do a clinical trial of this modality, to translate what I found in the lab into patients. So the clinical trial was designed in patients initially with recurrent malignant glioma. We confirm this by biopsy, we would not take out the tumor, we would just inject the virus and we injected several doses of the virus in a dose escalation fashion and then try to follow patients, for the occurrence of toxicity. Now this shows all the steps, the marathon you have to go through, to get something like this into humans. So really, the first time we published on this paper was 2005. We met with the FDA and we finally came up with a plan to basically do a final toxicology in mice and then we finally filed what's called an investigation, new drug approval. We had to then do something more, but finally this got approved by the FDA and we enrolled the first patient in 2017, okay? So this was a timeframe and this is actually when I sent off the IND to the FDA. This was back in 2015, 2016. And at that time, you still had to send them paper copies, this is about 5,000 pages and whatever, all the preclinical data on the virus and all the studies that we had to do and now, luckily, the FDA takes takes electronic documents, but that just shows you the amount of work, that need one needs to do. Because we were worried about the safety of this virus, this was again, they expressed this gene that causes herpes encephalitis, we wanted to make sure when we do stereotactic injection, that we really were in the tumor. So the initial phase of the trial, were done in the intraoperative MRI, using this ClearPoint MRI compatible system. And we use this tapered needle device, so that you limit reflux as you inject this and we injected the virus in the patient's tumors over five minutes or so. And this shows an intraoperative MRI reconstruction of a patient undergoing the injection. My postdoc colored the tumor here in purple, but if I take out the coloring here, you can see the brain. This was an occipital injection, to get to this temporal tumor and this is the end of the needle, where we're injecting the virus at this one location, the tumor and you can follow this with the intraoperative MRI. So we started this trial in 2017 and we actually treated a total of 51 patients, from 2017 until February of 2022, over a period of five years and these are all the patients that were enrolled in the trial. And then at that point, we saw the Japanese paper and we decided that we really wanted to do repeat dosing, up to six doses into the patient's tumors and Japan they were able to do that, 'cause everything is covered by their nationalized health insurance. But The States, repeat dosing is not covered by insurance. Each one of those procedures is with research procedure and each one of these is in the OR, so they're very, very expensive. So we really went out, even though we wanted to do this a long time, even before the Japanese paper, we just did not have the financial ability to do that, until Breakthrough Cancer, which is this large philanthropic donation, that was given to four cancer centers in the United States, including MIT came through and they decided to basically fund this trial, to do repeat dosing injections and at the same time, we can get serial biopsies, so we can actually analyze the tumor tissue longitudinally as we inject this virus and we're doing what's called single cell RNA sequencing, as well as other sophisticated technologies, to look at what's happening in tumor micro-environment, as you give this virus. So for the first two phases of trial, right here, and this is the data that's the most mature, this is the median overall survival. Again single dose in recurrent high-grade gliomas. The eligibility criteria for this were pretty liberal. We took first, second, third, or fourth recurrence. We took multifocal glioblastomas, we took glioblastomas that were in deep thalamic areas. Some of them that were bihemispheric, like going from one frontal lobe to another frontal lobe to the corpus callosum. So it was fairly liberal criteria. And so in spite of that, you know, the survival was fairly encouraging, it was about 11.7 months. These are all IDH wild-type gliomas. And again the expected survival, especially considering that about one third of these patients had really bad imaging characteristics is six to nine months. So they seem to be fairly encouraging survival. And let me just show you one example of a patient that did great. Here's a patient with a glioblastoma, that was initially recepted by me in the right frontal lobe and then it recurred after chemoradiation. You can see there's a reoccurrence here, but there's also a second recurrence. So this patient has a multifocal GBM, he has two GBM foci. So we took him to surgery and just injected one of them, the new focus and actually, you see the bubble of the injection in each opt MRI, right there. And then over the period of a year, you can see both of these tumors disappear. They kind of disappear, not just the one that we injected. Plus the one that was not injected, suggests there was an abscopal, or vaccination-like effect. Here's another patient, who did really well and this is another video, with the patient's family's consent.

- [Presenter] Meet Susan, a delightful, vibrant woman, who enjoyed life to the fullest. In December 2017, she started to experience mild headaches. Six weeks later, when driving home one day, she had a seizure and was transported to the emergency room, where it was quickly discovered that she had a brain tumor. Easily identified as glioblastoma, due to its unique presentation on imaging.

- She had this tumor this glioblastoma, that had grown in her temporal lobe and she initially had standard of care treatment. The standard of care treatment involves resecting this surgically and then she underwent radiation and chemotherapy.

- The treatments after the surgery were much more difficult for her to recover from. The chemotherapy especially was just, it decimated her. She is a very, or was a very active and vivacious person and when she was on the chemotherapy, she was just so sick and so nauseous all the time.

- [Presenter] After the recurrence, the patient was offered a new gene therapy, through a phase one clinical study, that was invented by Dr. Chiocca. The oncolytic virus therapy is delivered using an inter-operative MRI and is engineered to be tumor selected. The virus infects, fights and destroys the tumor cells. Initially she did well, but within 90 days, it looked like the tumor grew back for a third time. It was decided to go back to surgery. What was discovered, was what they thought were tumor cells, was the immune or T Cells attacking the tumor. Susan remained tumor free for two years and enjoyed life with her children and grandchildren.

- And after we resected this tumor, she actually did really well. For more than two years, she was tumor free, went back to work full time, traveled with her husband and unfortunately, she passed away, for reasons other than her tumor, totally unexpectedly.

- She was not sick all of the time. She continued doing a lot of the things she enjoys to do and she felt great. You know, she needed to rest after her infusions, which was usually every three weeks, is when she would get those, but they went on several cruises. It was wonderful. It was exactly what she wanted for her quality of life. It was perfect. So having that extension in life, but also the quality of life, that allowed her to come and travel multiple times, on her own, with family, go on cruises, like, she got to live her life, almost as normal as she could have wanted.

- So it's always important to see how patients are doing on trial and I gave you two examples of patients that did really well on this trial, although a lot of patients did not. Clearly, this is glioblastoma occurrence, so some patients do recur, even after this treatment. So the most important part of this trial, outside of the evaluation of the agent, to me is the science and just like what I showed you with the IL-12 gene therapy, we've really urged or prided ourselves, to try to get tumor back, whenever there is the potential possibility that that tumor is recurring, after the oncolytic viral injection, and this shows you in the timeframe, where you have the patient before treatment, at the time of surgery, they get an injection, the virus and then sometime after surgery and this was anywhere between 30 days, all the way out to almost four years after surgery, there's some evidence that the tumor is coming back and really endeavored to try to get craniotomies on those patients, to get the tumor back, so that we could query the tissue, at least with these two time points. And in fact, out of the first 41 patients, we end up getting back 32 of those tumors. So we now had this large trove of tumors that have been treated, that we could query, with all these analysis shown here. We also have blood longitudinally collected clearly during the course of this treatment. And so, we have analyzed a lot of these tumors and the paper is under re-review. Hopefully it'll get published fairly soon or we get accepted fairly soon. Because we did a lot of transcriptomic TC receptor analysis, to see what the T cell receptors are or what we're going to call T cell chrono types in the tumor, cytokines and other things that are relevant to do with the immune system and its interaction with this tumor. But I'm going to show you just two pieces of science from this trial and really the first question is, you know, we're injecting this oncolytic virus, so the first question we ask is, does the virus persist? You know, say it is supposed to replicate and diffuse by distributing the tumor, does that actually happen? And the second question is, can you by immuno-chemistry, see whether there's more CD8, CD4 and B cells in the GBM, because that's what this virus is supposed to do, supposed to get more T cells in there. So for the first question, I'm just gonna show you a couple of patients and this is a patient that had this ugly looking, motor strip, GBM, again, not a patient that's typically eligible for a trial, but we did inject this person with the virus and then six weeks later, you know, this is just becoming large and necrotic, she's becoming very symptomatic. So I did take her back to surgery, to try to get as much of this out. Initially, this area of necrosis that you see here is just full of herpes antigen. So there seems to be a SAR toxic response in this tumor, caused by the herpes virus. Here's a second patient, again, recurrent right frontal GBM. You could have taken this out by surgery, but you really did not wanna do that. So we did this minimally invasive injection. You can see intraoperatively in the MRI scan, this is where the needle is, the little bubble where the virus has been injected and he does well for a while, but then at nine months, there seems to be this large necrotic area that's happening in the right frontal layer, where we injected it. But there also seems to be some tumor escaping into corpus callosum as well as pre-matriculate. So we took them to surgery, to recept this and again, even nine months later, you can see that these necrotic areas are full of herpes antigen, but it's also full of CD8 T cells. There are a lot of T cells here and they're still trying to fight off this tumor. So again, the range is, we've been able to find the virus, even up to 801 days after injection. And the second question, and these are just a few more slides, is do you see more evidence of lymphocytes than B cells in these tumors? So these are quantitative analysis, carried out by Keith Ligan, together with Jared Woods and Isaac Solomon, who's really our neuro pathologist at trial and with the help of Candel Therapeutics, who now has licensed the virus. But here we're looking at these large necrotic areas that sometimes have herpes antigen and what you see, they're surrounded by CD4 and CD8 T cells, as well as CD20 B cells. But these cells are not just found in the areas of virus. You also find them far away, particularly in perivascular areas, as T cells are trying to get into the tumor. And you can see, that not only they're all CD8+ T cells in there, right? And then you can quantify this, so out of the 41 initial patients, we had 29 tumors where we did this and 21 out 29 tumors showed more T cells, highly statistically significant after injection. 24 out of 29 tumors showed more CD4+ T cells, highly statistically significant and in some patients, we see more B cells going up. But when you look at the complete collection of patients, it was not significant. We started doing some of the so-called transcriptomic analysis, this is another piece of data. So here we're looking at the transcripts that are being expressed in these tumors. And so you have all these signatures and what we're looking at, is what happens if you have a transcriptomic signature of T cells in the tumor. And you can see if you have more transcriptomes, that are usually due to T cells, these are the patients that survive the longest, compared to ones that have less. You can look at whether these T cells are effector cells. Effector cells mean these T cells are active, they're not exhausted. And again, if that is the case, if you have these transcriptomes, these are the patients that survive longer, compared to controls. And you can also look at checkpoint inhibition and again, if you have signals that have to do with checkpoint inhibition, these are the patients that survive longest, compared to the controls. You can also look at what's called T cell chrono types. These are T cell transcript, particularly transcripts, that's called VDJ chains. These are what the T cell receptor is made of and the more transcripts you have, the longer you survive. These are the patients in red, GBS specific mortality and if you have more transcripts elevated, you survive longer, than if you have less transcripts. So in summary, and I'm done here, is we can show the treatment with the CAN-3110 virus is well tolerated. We don't see those limiting toxicities. We can find the virus, by just looking immuno-chemically at the herpes antigen. We see evidence of replication, in almost half of the post-treatment samples. We see significant increase in CD4+, CD8+ T cells, after this virus. And we also, evidence for increased signatures and increase of these T cell receptor transcripts in longer survivors. And we're now in the course of doing this multiple repeat injection trial. We've done three patients out of 12 so far in a multi-situational fashion and so far, the first three patients have done well. So I have to thank, it takes a village to do these trials, both the preclinical, clinical studies. I only have the time to really thank everybody. The people on the line here are the ones that did a lot of the work that I've showed you and so I thank you for your attention and hopefully this was clear. Thank you.

- Thank you so much. You know, herculean task to say the least. I mean those giant binders that you demonstrated, just probably is the tip of the iceberg in these immunotherapy, especially viral clinical trials, because just the regulatory pathway is so extremely challenging and it's always been probably one of the highest barriers to these trials. If I may ask you, what in your opinion is the highest barrier, regulatory wise or in general, in these trials are so well needed, but unfortunately are so difficult to perform?

- Yeah, I mean, I think anytime you have a new drug or a new route of administration, you need FDA approval through an IND. There's just no, it's illegal. It's a law. So if you don't do that, you break a law, so that's a felony. So you really need to do that. And so, that's a big component. The FDA's main interest is safety, right? And you also, if you have a therapy that you wanna test, you need to make sure that the first patient you treat does not, basically, have a really bad side effect. And so really, the entire process of getting to an IND is to design a clinical trial, that has clear cut objectives. I always think that's the easiest part for a clinical trial. That's not a big deal. But the complicated part, that's really expensive, is to come up with your biologic agent or drug product, all the chemistry, the manufacturing, you know, you can do stuff in your lab, but if you scale it up, the scaling up process is a big deal. It's a lot of work, it's a big deal. You need to have experts to do this. You need to have people that know how to do this, and you know, you need to make sure that what you're giving patients does not have toxic compounds in it, doesn't have things that potentially could give side effects and finally, the other complicated thing that can be done is, the so-called bio distribution study of toxicology studying mice. And there, all you're trying to do, is find a dose that's well tolerated by mice, 'cause that's the dose that's gonna dictate what the first dose is, that you're gonna give patients and there's some rules around that, probably too complex to go through it right now, but I have to tell people about it. So that's a big burden. But you know, it's really meant to keep patients safe, even patients with terrible diseases like GBM. The FDA is a little bit more lenient with GBM. For example, they don't require two animal species, they just require one animal species. And so, there's some leniency there, but again, the regulatory burden is there and there's no way around it. And then, there's institutional regulatory burdens, right? So you have to go through an IRB, you have to have a clinical trial unit and help you do this 'cause the rules and regulations are pretty complex.

- I would say, you know, they're so extremely complex and in these patients who don't have many options and only have nine months to live and so, there has to be other ways. We're not trying to place the patients at risk. But as you know, this is a disease, that is a high risk, high reward solution. It requires a solution that has a high risk and high reward and it will not be a solution that is low risk, high reward, it just never works that way in the world. So I know they have designed these zero phase, zero trials, but it seems to me, that for us to make any major progress, there has to be a certain amount of efficient process to take new treatments to the patient and be able to try them and then revise it and, you know, change things and improvise, in very short intervals, with ways that are efficient enough regulatory-wise to do. Don't you agree?

- Yeah. And people have thought about that. So for example, doing drug repurposing, drugs that are already FDA approved, using them in GBM patients, that's easy, you don't have to get an IND, it's just drug repurposing. Especially if you give it systemically orally, 'cause again, you're not changing something about the drug or how you give it. People have designed these, what are called, Gainsight or AGILE trials. So these are trials where, basically, they're ongoing all the time, they have a control group that's always accruing and then you accrue drugs to your randomized groups and you can just add them on and their computer algorithm, that basically, if you have a pre-specified endpoint for example, could be progression, survival or whatever expression of some marker, if that drug seems to not meet those, the computerized, randomized program tends to not accrue patients to that arm, but to a newer arm of some other drug. And some of those trials are accruing patients faster because again, you don't have to put in a new IRB every time, so like an ongoing IRB protocol, that keeps ongoing, all the time and even with the FDA, it's always just an ongoing, you just add the arms to each one to the trials. So it's ongoing trials and those are getting published now and they seem to be, you know, they have an ability to evaluate drugs faster. The problem for us as neurosurgeons, is that those are neuro-oncology trials, you know, systemic drugs, chemotherapy drugs. For the biologics and the vaccines, especially if you give anything during surgery, those are difficult trials to accrue to like that and again, it still requires getting IND and doing the phase one initially, et cetera. But at least you are involved as a neurosurgeon, so I think that's the advantage and you can do this as part of your treatment, piggyback on a standard of care. Recurrent glioblastoma should be removed and you can do, also your clinical trial at the same time.

- Well, yeah, anyways, I commend you, for your incredible herculean task. Very few people in neurosurgery have done what you have done, with these regulatory barriers, with inertia, with the effort required to conduct these trials. As you know, overall pathways are not gonna cross the blood brain barrier, repurposed drugs have a low chance of getting to the brain tumor and so we require something much more innovative. What you have done, directly penetrating the brain, delivering the treatment and hoping what would happen. So I just hope that in the near future, there will be pathways to do all of them more efficiently, knowing that these patients have very limited access to treatment or options. So again, I wanna thank you, you know, incredible work, something very few people have achieved with what you have done in neurosurgery and thank you.

- Thank you, Aaron.

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